Molecular forms and kinetic properties of malate dehydrogenase and glutamate oxalacetate transaminase in Glenlea and Kharkov wheat cultivars

Abstract

Properties of crude extracts and partially purified mitochondrial (mt) fractions of NAD malate dehydrogenase (MDH) and glutamate oxalacetate transaminase (GOT) from spring (Glenlea) and winter (Kharkov) wheat cultivars were investigated. Plants were acclimated under controlled conditions at three different temperatures: 5/10°C, 15/25°C and 25/28°C. Both enzymes were more thermostable in mt than in crude extracts. The mt-MDH fraction, but not the unfractionated enzyme, of Glenlea plants was more thermostable than that of Kharkov plants and, for both cultivars, there was a tendency for a higher thermostability of the enzyme in plants acclimated to 25–28°C. For GOT no differences between cultivars were observed. There was a tendency for increased thermostability of GOT extracted from plants acclimated to 25–28°C. Apparent activation energies ( Q 20 10 °C ) of crude and mt-MDH of Glenlea plants acclimated to 5–10 and 25–28°C were significantly lower than those of Kharkov plants. E a values for GOT were very similar for both cultivars and were not modified by acclimation. Little difference between the two cultivars or between thermoperiods was observed for K m and V max/ K m ratios of both enzymes. For MDH, the apparent K m for oxalacetic acid increased with temperature. At 35°C, K m values were significantly higher and V max/ K m ratios lower in Kharkov as compared to Glenlea plants. For GOT, the K m for α-oxoglutaric acid did not vary significantly over the 10–35°C assay temperature range. Activities of both enzymes per g fresh leaf weight were significantly higher in Kharkov as compared to Glenlea plants acclimated to 5–10°C and were significantly higher in plants of both cultivars acclimated to this cold thermoperiod. No differences in the number of isozymes of MDH, consisting of one mitochondrial and four cytosol isozymes, or in their electrophoretic mobility in polyacrylamide gels were found between the cultivars. GOT electrophoretic patterns for both cultivars were identical consisting of three fast migrating mitochondrial isozymes and two groups of three cytosol isozymes.